Michitsune Arita

Expression of ACE and ACE2 in Individuals With Diabetic Kidney Disease and Healthy Controls

BACKGROUND Angiotensin-converting enzyme (ACE) 2 (ACE2) is expressed mainly in the heart and kidney and forms angiotensin-1-7 from angiotensin II. ACE2 might act in a counterregulatory manner to ACE. There is little information about renal ACE and ACE2 expression in human diabetic nephropathy. STUDY DESIGN Cross-sectional study. SETTING & PARTICIPANTS Kidney tissue from 20 patients with type 2 diabetes and overt nephropathy and 20 healthy kidney donors. PREDICTOR Diabetes status. OUTCOMES & MEASUREMENTS Renal expression of ACE and ACE2 assessed by means of immunohistochemistry and in situ hybridization. Correlation between ACE and ACE2 expression and levels of various biochemical parameters. RESULTS Decreased ACE2 and increased ACE expression in both the tubulointerstitium and glomeruli resulted in a significant (P < 0.001) increase in ACE/ACE2 ratio in patients with diabetes with overt nephropathy compared with controls, although ACE messenger RNA in the tubulointerstitium did not significantly increase. ACE/ACE2 ratio correlated positively with values for mean blood pressure, fasting blood glucose, serum creatinine, proteinuria, and hemoglobin A(1c) and inversely with estimated glomerular filtration rate (P < 0.001). LIMITATIONS Inclusion of small number of human renal biopsy specimens with structural distortion of cortical tissue. CONCLUSIONS The high ACE/ACE2 ratio in kidneys of patients with type 2 diabetes with overt nephropathy may contribute to renal injury.

Genetic Instability Caused by Loss of MutS Homologue 3 in Human Colorectal Cancer

Microsatellite instability (MSI) is a hallmark of mismatch repair (MMR) deficiency. High levels of MSI at mononucleotide and dinucleotide repeats in colorectal cancer (CRC) are attributed to inactivation of the MMR genes, hMLH1 and hMSH2. CRC with low levels of MSI (MSI-L) exists; however, its molecular basis is unclear. There is another type of MSI--elevated microsatellite alterations at selected tetranucleotide repeats (EMAST)--where loci containing [AAAG](n) or [ATAG](n) repeats are unstable. EMAST is frequent in non-CRCs; however, the incidence of EMAST and its cause in CRC is not known. Here, we report that MutS homologue 3 (MSH3) knockdown or MSH3-deficient cells exhibit the EMAST phenotype and low levels of mutations at dinucleotide repeats. About 60% of 117 sporadic CRC cases exhibit EMAST. All of the cases defined as MSI-H (16 cases) exhibited high levels of EMAST. Among 101 non-MSI-H cases, all 19 cases of MSI-L and 35 of 82 cases of MSS exhibited EMAST. Although non-MSI-H CRC tissues contained MSH3-negative tumor cells ranging from 2% to 50% of the total tumor cell population, the tissues exhibiting EMAST contained more MSH3-negative cells (average, 31.5%) than did the tissues not exhibiting EMAST (8.4%). Taken together, our results support the concept that MSH3 deficiency causes EMAST or EMAST with low levels of MSI at loci with dinucleotide repeats in CRC.

Urinary angiotensin-converting enzyme 2 in patients with CKD.

Aim:  Angiotensin‐converting enzyme 2 (ACE2) is a type I membrane protein that antagonizes the action of angiotensin II. Because of the need for invasive kidney biopsy, little is known about the role of renal ACE2 in human kidney diseases. The authors studied if urinary ACE2 could provide a novel clue to renal ACE2 in chronic kidney disease (CKD).

Increased ACE and decreased ACE2 expression in kidneys from patients with IgA nephropathy.

Background: Angiotensin-converting enzyme (ACE)2 forms angiotensin-1–7 which may protect kidney in a counterregulatory manner to angiotensin II. Recent studies revealed increased ACE and decreased ACE2 expression in kidneys of patients with diabetic nephropathy. However, these changes may not be specific for diabetic nephropathy. We studied ACE and ACE2 expression in patients with IgA nephropathy. Methods: Renal ACE and ACE2 expression was assessed by immunohistochemistry and in situ hybridization in 30 patients with IgA nephropathy and 21 healthy controls. Correlation between ACE and ACE2 expression and levels of various biochemical parameters was also assessed. Gene expression was also assessed in minimal change nephrotic syndrome (MCNS) and membranous nephropathy (MN) as disease controls. Results: Reduced ACE2 expression (p < 0.01) and increased ACE expression in glomeruli (p < 0.001), and reduced ACE2 expression in tubulointerstitium (p < 0.001) were observed in patients with IgA nephropathy compared to healthy controls, although the changes in ACE2 mRNA were not statistically significant. Reduced renal ACE2 expression was also found in MN but not in MCNS. Correlation between renal ACE and ACE2 expression and proteinuria was not observed in IgA nephropathy. Conclusion: IgA nephropathy is associated with increased ACE and decreased ACE2 expression in kidneys, as in diabetic nephropathy.

Antifibrotic response of cardiac fibroblasts in hypertensive hearts through enhanced TIMP-1 expression by basic fibroblast growth factor.

BACKGROUND Cardiac fibroblasts (CFs) play a pivotal role in the development of myocardial fibrosis. We previously demonstrated that direct injection of basic fibroblast growth factor (bFGF) into the hypertensive Dahl salt-sensitive (DS) rat heart prevented systolic dysfunction and left ventricular dilation effectively. However, the precise role played by bFGF in fibrotic response of CFs remains unclear. We suggested potential effects of bFGF on the fibrotic response of CFs in vitro. METHODS AND RESULTS Histopathologic assessment of cardiac fibrosis demonstrated a marked decline in the extent of perivascular and interstitial fibrosis in bFGF-injected hypertensive DS rat hearts. CFs harvested from the hearts of noninjected DS rats demonstrated a significantly increased messenger RNA (mRNA) expression of matrix metalloproteinase (MMP)-2, MMP-9, and both collagen I and III. In contrast, bFGF treatment in the CFs induced a marked increase in tissue inhibitor of MMP (TIMP)-1 expression and a marked decline in MMP-9 activation. bFGF also induced a decline in α-smooth muscle actin and collagen I and III mRNA expression in the CFs accompanied by inhibited differentiation of CFs into myofibroblasts. Small interfering RNA targeting FGF receptor 1 confirmed a specific interference of the mRNA expression changes elicited by bFGF. In vivo examination confirmed many TIMP-1-positive CFs in perivascular spaces of bFGF-injected hearts. CONCLUSIONS Up-regulated TIMP-1 expression and down-regulated MMP-9 activation by bFGF in CFs could prevent excessive ECM degradation and collagen deposition in perivascular spaces effectively, leading to prevention of cardiac fibrosis during hypertensive heart failure. SUMMARY Cardiac fibroblasts (CFs) play a pivotal role in myocardial fibrosis. The precise role of CFs in fibrotic response played by growth factors remains unclear. Our results indicates that basic fibroblast growth factor could up-regulate TIMP-1 expression and down-regulate MMP-9 activation in CFs in perivascular spaces, leading to inhibited progression of cardiac fibrosis during hypertensive heart failure.

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer.

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.

Effects of New Peritoneal Dialysis Solutions, Pyridoxamine and AT1 Receptor Blocker, on TGF-β1 and VEGF Expression in Rat Peritoneal Mesothelial Cells

Background: Transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) are involved in peritoneal deterioration in continuous ambulatory peritoneal dialysis. The present study was designed to determine whether new peritoneal dialysis solutions (PDS), pyridoxamine (advanced glycation end products (AGE) inhibitor) or AT1 receptor blocker (ARB), affect the expression of VEGF and TGF-β1 in rat peritoneal mesothelial cells (RPMC). Methods: RPMC were stimulated by phosphate-buffered saline (PBS) as control, Dianeal 1.5% (D 1.5%), Dianeal 2.5% (D 2.5%), Dianeal 4.25% (D 4.25%), Dianeal N 1.5% (N 1.5%), Dianeal N 2.5% (N 2.5%) or Extraneal (Ex). In co-incubation experiments, RPMC were stimulated with N 2.5% including pyridoxamine or olmesartan (ARB). VEGF and TGF-β1 protein and mRNA expression in RPMC were analyzed by ELISA and RT-PCR. Results: Glucose-containing PDS, even N 2.5% diluted twofold with M199 (which contains 1.25% glucose), increased VEGF and TGF-β1 expression in RPMC (p < 0.05). Ex did not inhibit VEGF expression and did not inhibit TGF- β1 expression after 24 h in RPMC. Pyridoxamine and ARB significantly reduced N 2.5%-induced VEGF and TGF-β1 protein and mRNA expression in RPMC (p < 0.01). Conclusions: Neither new pH-neutral, lactate-buffered, low-GDP, two-chamber bag PDS, nor 7.5% icodextrin PDS alone satisfactorily inhibited VEGF and TGF-β1 overproduction in RPMC, but ARB or pyridoxamine effectively inhibited glucose-containing PDS (N 2.5%)-induced overproduction.

Effluent markers related to epithelial mesenchymal transition with adjusted values for effluent cancer antigen 125 in peritoneal dialysis patients.

Objectives. Epithelial mesenchymal transition (EMT) is important for peritoneal deterioration. We evaluated the association between peritoneal solute transport rate (PSTR) and effluent markers related to EMT with adjusted values for effluent cancer antigen 125 (CA125). Methods. One hundred five incident peritoneal dialysis (PD) patients on PD for 25 (12–68) months with biocompatible solutions were included in the study. Fast peritoneal equilibration test was used to evaluate PSTR. Effluent hepatocyte growth factor (HGF), bone morphogenic protein-7 (BMP-7), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and CA125 at 4 h were measured. Results. Patients with dialysate/plasma creatinine ≧0.82 showed significantly higher effluent HGF (240 versus 133 pg/mL, P < .001), VEGF, IL-6, and IL6/CA125 levels than the others but no significant differences in effluent HGF/CA125, BMP-7, and BMP7/CA125 were observed. Conclusion. Increase in the effluent HGF levels as a compensatory mechanism is a marker of peritoneal deterioration, but controversy remains regarding adjusted value for CA125.

ACE2- Ang 1-7-MAS Axis in Human Diabetic Nephropathy

The angiotensin-converting enzyme 2 (ACE2)- angiotensin-(1-7) [Ang (1-7)]-MAS receptor (MAS) axis might act as a counter-regulatory system against the angiotensin-converting enzyme (ACE)- angiotensin II (Ang II)-AT1 receptor (AT1) axis. We studied renal ACE/ACE2-Ang 1-7-MAS expression in human diabetic nephropathy. The subjects were 17 diabetic nephropathy (DN) patients, 17 healthy kidney donors, and 11 minimal change nephrotic syndrome (MCNS) patients as disease controls. Double immunofluorescent staining of kidney sections for ACE and ACE2, Ang 1-7 and ACE2, or MAS and ACE2 was performed, and the results were observed by confocal microscopy. For MAS, immunostaining, in situ hybridization, and RT-PCR were also performed. The percentage area that was positively immunostained for MAS and the intensity of the staining were evaluated by computerized imaging analysis. The median serum creatinine values of the DN patients, MCNS patients, and controls were 1.1 mg/dl, 0.8 mg/dl, and 0.8 mg/dl, respectively, and the median proteinuria values of the DN and MCNS patients were 3.7 g/day and 6.8 g/day, respectively. ACE2 was mainly detected in the proximal tubules, but was also found in the glomeruli in all subjects. ACE2 co-localized with ACE, Ang 1-7, and MAS in the proximal tubules. Compared with the other specimens, the diabetic patients’ proximal tubules displayed increased ACE and decreased ACE2 expression. Tubular Ang 1-7expression was downregulated in the DN patients compared with the controls and MCNS patients. Tubular MAS expression (density/pixel) was significantly (P<0.001) downregulated in the DN patients [7.33 (6.26-9.68)] compared with the controls [24.51 (18.06-34.56)] and MCNS patients [23.75 (20.11-25.52)]. Conclusions: The tubular ACE2-Ang 1-7-MAS axis is downregulated in human diabetic nephropathy patients compared with healthy controls and MCNS patients

Amino acid analyses of the exosome-eluted fractions from human serum by HPLC with fluorescence detection

Objectives Amino acid levels in serum or plasma are used for early detection and diagnosis of several diseases. The objective of this study was to analyze amino acid levels in serum exosomes, which have not been previously reported. Design and methods We investigated the amino acid composition of exosomes from human serum using HPLC with fluorescence detection. Results The composition ratios of His, Arg, Glu, Cys-Cys, Lys, and Tyr were significantly increased in the exosomes compared with those in the corresponding native serum. d-Ser, an endogenous co-agonist of the N-methyl-d-aspartate receptor, was also enriched in the exosome-eluted fraction. Conclusions Our results suggest that certain amino acids are enriched in the exosome-eluted fraction from human serum. These differences could have future diagnostic potential.